There is an urgent need to discover more effective therapeutic regimens for fungal infections. Nosocomial infections caused by Candida albicans have a 50% mortality rate. Aspergillosis is a leading cause of death in organ transplant recipients, as well as patients suffering from cancer and auto-immune disorders. The annual cost of treating fungal infections is about $2.6 billion in the United States and is increasing due to the larger number of immunocompromised patients who suffer from these illnesses. The emergence of fungal drug resistance to widely used antifungals including triazoles and echinocandins further compromises the efficacy of the limited armamentarium of antifungal therapeutics. A number of in vitro and in vivo studies have established that molecules which inhibit the fungal protein, calcineurin, are highly synergistic with several important classes of antifungal therapeutics including triazoles and echinocandins. However, a great challenge with exploiting fungal calcineurin as a therapeutic target is the structural similarity to human calcineurin, and that inhibition of human calcineurin causes severe immunosuppression and toxicity. By exploiting recently developed chemistry to generate a variety of FK506 analogues, we have isolated a molecule which has equivalent or higher antifungal activity than FK506 and negligible immunosuppression. When combined with common antifungals, this molecule had excellent potency against drug resistant clinical isolates. Encouraged by these initial results, we wish to create a larger library of FK506 analogues in order to discover molecules that are non-toxic, highly antifungal, and synergistic with existing therapeutics. Our overall goal is to develop an effective combination therapy which can be applied to drug resistant fungal infections. Our aims are: Aim 1. Design and synthesis of a 1000 member focused library of FK506 analogues. Aim 2. Screen and select library for lack of immunosuppression. Aim 3. Screen and select library for antifungal effects when combined with established antifungal drugs. PUBLIC HEALTH RELEVANCE: Fungal infections represent a significant challenge due to the limited armamentarium available to treat diseases caused by fungi. Nosocomial infections caused by Candida albicans have a 50% mortality rate. Amplyx proposes a new combination therapy approach to treating drug resistant fungal infections via the design and synthesis of non-immunosuppressive molecules to inhibit fungal calcineurin that can be co-administered with existing antifungals.